The composition and structure of the extracellular matrix plays a central role in the patterning of the cardiovascular system. In addition to its developmental role, the extracellular matrix often initiates thrombus formation. Project 2 is focused on the fundamental mechanisms controlling the assembly of one component of the extracellular matrix, insoluble fibronectin fibrils. The studies will address intracellular mechanisms that control integrin functions essential for the matrix assembly process. In particular, this project will analyze the mechanisms response for "activation" of the high affinity form of integrin alpha5beta1 and for its physical linkage to the cytoskeleton. These aims will be accomplished by isolating cell lines with defects in cellular pathways required for activation of integrin alpha5beta1. Expression cDNA cloning will then identify Genes that complement those defects. An alternative strategy will employ enrichment of complemented radiation hybrids to identify chromosomal positions of complementing genes. The genes will then be further localized through use of sequence tagged sites, rescue by transfection with P1 clones and finally rescue by transfection of candidate cDNAs. Analysis of integrin-cytoskeleton linkage will employ novel recombinant structural mimics of the beta cytoplasmic domains and analyze their interactions with two actin binding proteins, filamin and talin. Interactive sites in the integrin and the cytoskeletal proteins will be mapped. This information will be used to test the hypothesis that the differential binding of these proteins to different integrins leads to integrin-specific functions. The studies will provide fundamental insight into the assembly of a critical component of the extracellular matrix of vascular cells.